Deletion of Switch 3 Results in an Archaeal RNA Polymerase That Is Defective in Transcript Elongation

Thomas J Santangelo,John N Reeve

doi:10.1074/jbc.m109.094565

Abstract

Switch 3 is a polypeptide loop conserved in all multisubunit DNA-dependent RNA polymerases (RNAPs) that extends into the main cleft of the RNAP and contacts each base in a nascent transcript as that base is released from the internal DNA-RNA hybrid. Plasmids have been constructed and transformed into Thermococcus kodakaraensis, which direct the constitutive synthesis of the archaeal RNAP subunit RpoB with an N-terminal His(6) tag and the Switch 3 loop either intact (wild-type) or deleted (DeltaS3). RNAPs containing these plasmid-encoded RpoB subunits were purified, and, in vitro, the absence of Switch 3 had no negative effects on transcription initiation or elongation complex stability but reduced the rate of transcript elongation. The defect in elongation occurred at every template position and increased the sensitivity of the archaeal RNAP to intrinsic termination. Comparing these properties and those reported for a bacterial RNAP lacking Switch 3 argues that this loop functions differently in the RNAPs from the two prokaryotic domains. The close structural homology of archaeal and eukaryotic RNAPs would predict that eukaryotic Switch 3 loops likely conform to the archaeal rather than bacterial functional paradigm.

Highlights

That archaeal and bacterial RNA polymerases (RNAPs) respond to different intrinsic termination signals [13,14,15, 18], it was of interest to determine whether the Switch 3 loop in an archaeal RNAP contributed to elongation complex stability and transcription termination
Loss of Switch 3 Does Not Affect Initiation but Reduces Elongation—For convenience, the RNAPs purified by Ni2ϩ affinity chromatography from lysates of T. kodakaraensis KW128 and KW128 are designated WT and ⌬S3, respectively
The presence of the comple- the Switch 3 loop from a bacterial RNAP resulted in an mentary strand, had no discernible positive or nega- enzyme that formed unstable elongation complexes that tive effect on the different elongation abilities of WT and ⌬S3 were readily disrupted by exposure to physiological salt con

Summary

Introduction

The template DNA (10 nM) was incubated with 40 nM RNAP, 80 nM TBP, 80 nM TFB1 or TFB2, 75 ␮M [32P]ApC in transcription buffer (20 mM Tris-HCl (pH 8), 250 mM KCl, 5 mM dithiothreitol, 5 mM MgCl2, and 7% glycerol) for 10 min at 85 °C to allow open complex formation. Purification of Elongation Complexes and Assay of NaCl and Heparin Sensitivity—In vitro transcription reaction mixtures that contained biotinylated template 442 [14], RNAP, TBP, TFB2, 75 ␮M [32P]ApC, 1 mM GTP, 1 mM UTP, and 50 ␮M ATP were incubated for 10 min at 85 °C.

Results

Conclusion